Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (H). However, the role of their conserved N-terminal sub-domain (H) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking H (rAΔH) and showed that the lethality of this mutant is reduced 3.3 × 10-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔH failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with I-BoNT/A (K = 0.46 nM). At a higher concentration, rAΔH did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within H reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that H makes additional contributions to productive internalization/translocation steps beyond binding to neurons.